1. Field of the Invention
The present invention relates to an apparatus and method for printing biomolecular droplets on a substrate, and more particularly, to an apparatus and method for printing biomolecular droplets having a small volume and diameter at a desired position.
2. Description of the Related Art
As a result of the research conducted during the Human Genome Project, there is an increasing need for methods of rapidly providing a large amount of genetic information for the diagnosis, treatment, and prevention of genetic disorders. Although the Sanger method of analyzing nucleotide sequences has been continuously improved through the development and automation of a polymerase chain reaction (“PCR”) method, in which deoxyribonucleic acid (“DNA”) molecules are duplicated, the Sanger method is still a complex, time consuming, labor intensive, and expensive technique which requires a lot of expertise to implement. Thus, analyzing a large number of genes using the Sanger method becomes prohibitive. As a result, new systems for analyzing nucleotide sequences are continuously being researched, and in the last few years, there have been advances in many fields relating to the manufacture and application of biochips.
A biochip is a biological microchip which includes a solid substrate made of, for example, silicon, surface-modified glass, polypropylene, or activated polyacrylamide. Biochips can be used to analyze gene developing patterns, genetic defects, protein distribution, or various kinds of reaction patterns when combined with biomolecules such as nucleic acids, proteins and cells.
If a target material to be analyzed is applied to a biochip, the target material hybridizes with probes immobilized on the biochip. The hybridization is optically or radiochemically detected and analyzed to identify the target material. For example, if a fragment of target DNA to be analyzed is applied to the DNA chip (or DNA microarray) on which probes are disposed, the target DNA complementarily hybridizes with the probes immobilized on the biochip. The hybridization is detected and analyzed using various detecting methods to identify the nucleotide sequence of the target DNA. This is known as sequencing by hybridization (“SBH”).
An example of a printing apparatus for manufacturing a biochip or a DNA microarray is disclosed in Korean Patent Application No. 2005-0040162. FIG. 1 is a schematic cross-sectional view of a printing apparatus 1 disclosed in the above reference for printing biomolecular droplets on a substrate using an electrohydrodynamic phenomenon. Referring to FIG. 1, the printing apparatus 1 includes: a first electric field forming electrode 4 which is needle-shaped, formed of a conductive material, is disposed vertically, and comprises an accommodating area 2 in which a biomolecular droplet, such droplets containing nucleic acids (e.g., probe DNA, RNA, PNA, and LNA), proteins (e.g., antigen and antibody), oligopeptides, eukaryotic cells (e.g., human cells, animal cells, and vegetable cells), viruses, and bacteria, is accommodated and a nozzle 3 formed on a bottom end of the accommodating area 2 through which the biomolecular droplet is discharged; a substrate 6 disposed below the first electric field forming electrode 4, and including a target surface 5 onto which the biomolecular droplets 10 ejected from the nozzle 3 of the first electric field forming electrode 4 are deposited; and a second electric field forming electrode 7 made of a conductive material, disposed below the first electric field forming electrode 4, and attached to the substrate 6. In addition, a voltage applying device 9 is connected to and applies a voltage to the first and second electric field forming electrodes 4 and 7 via an electrode lead wire 8.
As described above, in the printing device 1, when DC and AC voltages are simultaneously applied to the first and second electric field forming electrodes 4 and 7 by driving the voltage applying unit 9, an electric field is generated between the first and second electric field forming electrodes 4 and 7 as illustrated in FIG. 2. FIG. 2 is a schematic diagram of electric field distribution formed when a voltage is applied to the printing apparatus of FIG. 1. In FIG. 2, an electric force is generated from around the biomolecular droplet 10 towards the substrate 6 due to the interaction between the electric fields generated as described above. The biomolecular droplet has a free surface, and the atmosphere may have a dielectric constant gradient. Accordingly, the biomolecular droplet 10 suspended from the nozzle 3 is ejected onto the target surface 5 of the substrate 6 by the applied electric force.
However, in another embodiment the printing device 1 can form an electric field between the first electric field forming electrode 4 and the substrate 6 when the substrate 6 is made of a conductive material or in yet another embodiment the second electric field forming electrode 7 made of a conductive material may be attached to the substrate 6, and thus, an electro-hydrodynamic effect can be generated to eject the biomolecular droplet 10. Accordingly, the substrate 6 should be made of a conductive material or in the alternative, the surface of the substrate 6 should be conductive.
As illustrated in FIG. 2, the electric field generated between the first electric field forming electrode 4 and the second electric field forming electrode 7 may not be uniform, and thus the biomolecular droplet 10 may not be ejected onto a desired position of the target surface 5 of the substrate 6.
When the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 is less than a predetermined distance, an undesirable electric discharge (also known as a spark) can be generated. Since the electric discharge may change the biochemical characteristics, size, and volume of the biomolecular droplet 10, and the surface structure or characteristics of the substrate 6, the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 should be controlled to prevent the generation of an electric discharge. For example, when the substrate 6 is coated with polymethlymethacrylate (“PMMA”) and the coating thickness is 5 μm, the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 is held to more than 750 μm to prevent the generation of an electric discharge. The required distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 limits the device design. In addition, when the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 is too great, it is difficult for the biomolecular droplet 10 to be ejected onto a desired position of the target surface 5 of the substrate 6. Deposition of a sample onto a target is then a balance between avoiding electrical discharges and increasing positioning accuracy.
In order to solve such a problem in which it is difficult to eject the biomolecular droplets 10 onto a desired position of the target surface 5 of the substrate 6 while avoiding discharges, a ring-shaped electrode is introduced as a second electrode to form an electric field only within a ring. Thus, an apparatus as illustrated in FIG. 3 was developed. FIG. 3 is a schematic cross-sectional view of another conventional printing apparatus for printing biomolecular droplets on a substrate using an electrohydrodynamic phenomenon (Electric field driven jetting: an emerging approach for processing living cells, Biotechnol. J. 2006, 1, 86-94; Electric field driven jetting: Electrohydrodynamic Jet Processing: An Advanced Electric-Field-Driven Jetting Phenomenon for Processing Living Cells Small. 2006, 2, No. 2, 216-219; Electrohydrodynamic jetting of mouse neuronal cells, Biochemical journal, 2006 Jan. 4). Referring to FIG. 3, when biomolecular droplets are ejected out of an electric spray needle, which corresponds to a first electrode as described above, by an electric field formed with a ring-shaped electrode, which corresponds to a second electrode as described above, biomolecular droplets are ejected only within the ring-shaped electrode and reach the target surface. However, although biomolecular droplets are ejected into only within the ring-shaped electrode, the ring-shaped electrode must be separated from the electric spray needle by a predetermined distance in order to prevent an electrical discharge from being generated. Thus, the biomolecular droplets still can not be consistently ejected onto a desired position of a substrate.
To solve the problems of printing biomolecular droplets using an electrohydrodynamic effect, an apparatus 100a for printing biomolecular droplets as illustrated in FIG. 4 using an electric charge concentration effect was disclosed in Korean Patent Application No. 2005-74496. In the apparatus 100a, when an open circuit type voltage applying unit 60a simultaneously applies DC and AC voltages to an electric field forming electrode 20a after biomolecular droplets are first supplied to an accommodating area 22a, positive charges migrate into the biomolecular droplets 10a (not shown in FIG. 4, but shown in FIG. 5) which are suspended from a nozzle 23a, and thereby negative charges are induced along a target surface 31a in a substrate 30a which is grounded.
An electric field is formed between the positive charges and the negative charges as illustrated in FIG. 5. Accordingly, when positive charges migrate into the biomolecular droplets 10a, thereby inducing negative charges in a portion of the substrate 30a disposed opposite the biomolecular droplet 10a, a force is generated between the positive charges and the negative charges. Here, the negative charges are induced below the biomolecular droplet 10a, so that the force is concentrated on the bottom of the biomolecular droplet 10a. The biomolecular droplet 10a suspended from the nozzle 23a is ejected onto the substrate 30a due to the force as illustrated in the fourth photo of FIG. 6, and as illustrated in the schematic representation shown in FIG. 7, and is thus converted to an approximately jar shaped biomolecular droplet. A neck is formed in the jar-shaped biomolecular droplet 10a. 
Accordingly, when the biomolecular droplet 10a suspended from the nozzle 23a is ejected onto the substrate 30 and then formed as illustrated in FIG. 7, the positive charge in the biomolecular droplet 10a is cancelled by the negative charge formed on the substrate 30a, resulting in a reduction in force. That is, the force which causes the biomolecular droplet 10a suspended from the nozzle 23a to be ejected is decreased after the droplet 10a contacts the target area on the substrate. In addition, a surface tension A between the neck-shaped biomolecular droplet 10a and the substrate 30a, and a surface tension B between the neck-shaped biomolecular droplet 10a and the electric field forming electrode 20a act in directions opposite to each other as illustrated in FIG. 7. Accordingly, when the positive charge in the biomolecular droplet 10a is cancelled, the force is decreased and the surface tensions A and B also act opposite to each other. Thus, due to the force of the surface tensions, the biomolecular drop 10a is separated at the neck-shaped portion to become two biomolecular droplets.
Accordingly, the biomolecular droplets are deposited on the substrate 30a as illustrated in the last photo of FIG. 6. In the apparatus 100a, the substrate 30a is grounded. Thus, the substrate 30a can be made of any material. In addition, negative charges can be induced on a portion of the substrate 30a disposed opposite to the biomolecular droplet 10a by the positive charges in the biomolecular droplet 10a, and since a larger amount of positive charge is developed in the biomolecular droplet 10a as compared to when an electrohydrodynamic effect is used, the biomolecular droplet 10a can be ejected and deposited on a desired position of substrate 30a. 
In addition, a very high force acts so that the biomolecular droplet 10a can be printed with a smaller size and volume than those of the biomolecular droplet in the prior art. Also, the substrate 30a is grounded, thereby electric discharge is not generated unlike when an electrohydrodynamic effect is used as in the prior art. Accordingly, a distance between the electric field forming electrode 20a and the substrate 30a can be freely adjusted. That is, in the apparatus 100a for printing biomolecular droplets disclosed in Korean Patent Application No. 2005-74496, it is possible that biomolecular droplets having a small size and volume are printed on a desired position using an electric charge concentration effect.
However, to manufacture a biochip having a high density, a method of printing biomolecular droplets having an even smaller volume is required. In particular, in order to conduct research into the interaction of cells, including stem cells, the printing of biomolecular droplets having a volume as small as 6 or fewer cells per biomolecular droplet are required. The size of the required biomolecular droplet varies depending on a concentration of cells, but, for example, when 3% of a glycerol medium solution having a concentration of 3×106 cells/ml is to be printed, a biomolecular droplet having a diameter of 60 μm or less is required.
Therefore, a method of printing biomolecular droplets having a smaller volume and diameter using the device for printing biomolecular droplets disclosed in Korean Patent Application No. 2005-74496 is desired.